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RED CELLS

Loxosceles intermedia spider envenomation induces activation of an endogenous

metalloproteinase, resulting in cleavage of glycophorins from the erythrocyte
surface and facilitating complement-mediated lysis
Denise V. Tambourgi, B. Paul Morgan, Rute M. G. de Andrade, Fábio C. Magnoli, and Carmen W. van den Berg

Loxosceles is the most venomous spider
in Brazil, and envenomation causes der-
monecrosis and complement (C)-depen-
dent intravascular hemolysis. The au-
thors studied the mechanism of induction
of C-induced hemolysis. Purified Loxos-
celes toxins rendered human erythro-
cytes susceptible to lysis by human C but
did not have an effect on the E-bound
C-regulators DAF, CR1, or CD59. How-
ever, incubation with venom toxins
caused cleavage of glycophorin from the
erythrocyte (E) surface, facilitating C
activation and hemolysis. The results
suggest that glycophorin is an important
factor in the protection of E against
homologous C. Cleavage of glycophorin
(GP) A, GPB, and GPC occurred at sites
close to the membrane but could not be
accomplished using purified GPA and
purified toxins, demonstrating that cleav-
age was not an effect of a direct proteo-
lytic action of the Loxosceles toxins on
the glycophorins. Inhibition of the cleav-
age of glycophorins induced by Loxosce-
les venom was achieved with 1,10-
phenanthroline. The authors propose that
the sphingomyelinase activity of the tox-
ins induces activation of an endogenous
metalloproteinase, which then cleaves
glycophorins. They observed the transfer
of C-dependent hemolysis to other cells,
suggesting that the Loxosceles toxins
can act on multiple cells. This observa-
tion can explain the extent of hemolysis
observed in patients after envenomation.
Identification of the mechanism of induc-
tion of susceptibility to C-mediated lysis
after Loxosceles envenomation opens up
the possibility of the development of an
effective therapeutic strategy. (Blood.
2000;95:683-691)
r 2000 by The American Society of Hematology

Introduction
Envenomation by spiders of the genus Loxosceles, found in
temperate and tropical regions of North and South America, Africa,
and Europe, commonly results in impressive local necrotic skin
lesions and, more rarely, causes systemic effects, including pro-
found intravascular hemolysis .1-8 The predominant clinical sign is
a cutaneous reaction characterized by the appearance of necrosis
around the bite, resulting in ulceration. Healing of the ulcer often
requires months. The scale of these lesions is remarkable consider-
ing that the spider injects only a few tenths of a microliter of venom
containing no more than 30 µg protein. Mild systemic effects
induced by envenomation, such as fever, malaise, pruritus, and
exanthema are common, whereas intravascular hemolysis and
coagulation, sometimes accompanied by thrombocytopenia and
renal failure, occur in approximately 16% of those bitten.1-8
Loxosceles is the most poisonous spider in Brazil, and children who
have the more severe systemic effects after envenomation nearly
always die. At least 3 different Loxosceles species of medical
importance are known in Brazil (L. intermedia, L. gaucho, L.
laeta), and more than 1500 cases of envenomation by L. intermedia
alone are reported each year. In the United States, at least 6
Loxosceles species (including L. reclusa, the brown recluse) are
known to cause numerous incidents.4,6,8 Because of a lack of
understanding of the venom’s mechanism of action, effective
treatment is unavailable. Biochemical and functional characteriza-
tion of the active components in the venom may aid the develop-
ment of a suitable therapy.
We have recently identified and characterized the toxins from L.
intermedia venom that are responsible for all the local and systemic
effects induced by whole venom.9,10 Two highly homologous
proteins with Mr 35 kd were purified to homogeneity and were
shown to be endowed with sphingomyelinase activity. These
proteins, termed P1 and P2, induced dermonecrosis in experimental
animals and rendered human erythrocytes susceptible to lysis by C
in vitro. In a mouse model of Loxosceles envenomation,11 we
showed that the toxins also induced intravascular hemolysis and
provoked a cytokine response resembling that seen in endotoxic
shock.8 The spider toxins P1 and P2, which probably originated by
gene duplication, showed a high level of homology with two other
molecules in the spider venom. The latter molecules, named P3 and
P4—despite the high degree of homology in N-terminal amino acid
sequence with P1 and P2—were ineffective in all assays of
activity.10 The mechanism by which a single molecule displaying
only sphingomyelinase activity can induce such a wide variety of
local and systemic effects is the subject of this study.
We have shown that the E lysis induced by venom is dependent
on the activation of C by an alternative pathway.9 The toxins P1 and
P2, unlike some other sphingomyelinases, do not directly induce E
lysis, but they do render cells susceptible to C lysis. Acquisition of

From the Laboratório de Imunoquı́mica, Instituto Butantan, São Paulo, Brazil;
and the Departments of Medical Biochemistry and of Pharmacology, Toxicol-
ogy and Therapeutics, University of Wales, College of Medicine, Cardiff, United
Kingdom.
Submitted July 12, 1999; accepted September 13, 1999.
Supported by FAPESP, CNPq and by a Senior Fellowship from The Wellcome
Trust.
Reprints: Denise V. Tambourgi, Laboratório de Imunoquı́mica, Instituto Butan-
tan, Avenida Prof. Vital Brazil, 1500, CEP 05508-900 São Paulo, Brazil; e-mail:
butlim@eu.ansp.br.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
r 2000 by The American Society of Hematology

BLOOD, 15 JANUARY 2000 • VOLUME 95, NUMBER 2 683

 From www.bloodjournal.org by guest on May 25, 2017. For personal use only.
684 TAMBOURGI et al
C-activating capacity or loss of C regulation by treated E leads to
the deposition of C fragments, including C3b and factor B,
C3-convertase assembly, and membrane attack complex (MAC)
formation, with hemolysis as the final outcome.9 Erythrocytes are
protected against lysis by their own C by a number of specialized
regulators of C (CR).12,13 A high level of expression of the
regulators of the C3/C5 convertases decay accelerating factor
(DAF) and C receptor 1 (CR1) and the regulator of the MAC,
CD59 ensures the survival of E in vivo. The importance of CR is
illustrated by the spontaneous occurrence of hemolysis in patients
with paroxysmal nocturnal hemoglobinuria (PNH).14-17 In PNH,
because of a clonal defect in the anchorage of glycosyl phosphati-
dylinositol-anchored molecules, DAF and CD59 are not expressed
on the surface of erythrocytes.14 Although CR1 is expressed in
normal amounts, DAF and CD59 deficiencies render these PNH E
susceptible to C-dependent lysis. The importance of DAF, and of
CD59 in particular, in the protection against C is also demonstrated
by the sensitivity to C lysis of human E after the blocking of DAF
and CD59 by monoclonal antibodies (mAb) or biotin–avidin
cross-linking.18 Other factors have been shown to protect erythro-
cytes against lysis by homologous C, including surface carbohy-
drates.19-25 Glycophorins, heavily glycosylated proteins that are the
most abundantly expressed molecules on E, have been shown to act
as inhibitors of C-deposition, a consequence of the presence of high
amounts of sialic acid in the structures.20,21,22 Removal of sialic
acid by neuraminidase results in an enhanced susceptibility of E to
C lysis as a consequence of the reduction of binding of factor H
(fH; cofactor for factor I in the degradation of C3b) to surface-
bound C3b.23,24 Furthermore, it has been shown that alteration in
the lipid composition of membranes can affect the susceptibility to
C.25
The aim of this study was to elucidate the precise mechanism by
which P1 and P2 toxins from Loxosceles intermedia venom induce
C-susceptibility in E. Human E, treated with toxins, were examined
for the expression of CR, DAF, CD59, and CR1. No change in
expression was observed, which eliminated the possibility that
C-susceptibility was induced by the removal of these proteins by
the spider toxins. However, toxin treatment of E caused cleavage of
the extracellular portions of glycophorins (GP) A, GPB, and GPC.
As a consequence, C3b deposition was enhanced and was followed
by the activation of terminal pathway and C5b-C9 lytic complex
formation, with hemolysis as the final outcome. Indeed, the
removal of sialic acid by neuraminidase had the same effect on C
susceptibility as treatment of E with the spider toxins.
Materials and methods
Chemicals, reagents, and buffers
Tween 20, bovine serum albumin (BSA), human GPA, neuraminidase,
dimethylsulfoxide, 1,10 phenanthroline, and phenylmethylsulfonyl fluoride
(PMSF) were purchased from Sigma (St. Louis, MO). Calcein-AM was
from Molecular Probes (Cambridge, UK). Supersignal chemiluminescent
developer was from Pierce Chemical (Rockford, IL). Buffers were veronal-
buffered saline (VBS21), pH 7.4, containing 10 mmol/L Na barbitone, 0.15
mmol/L CaCl2, and 0.5 mmol/L MgCl2; PBS containing 10 mmol/L Na
phosphate and 150 mmol/L NaCl, pH 7.2; and FACS buffer containing 1%
PBS, 0.01% BSA, and sodium azide.
Antibodies
The following mAbs were all from International Blood Group Reference
Laboratory (IBGRL, Bristol, UK): mAb against DAF (Bric216), against
BLOOD, 15 JANUARY 2000 • VOLUME 95, NUMBER 2
CD59 (Bric229), against GPA (Bric256, recognize extracellular part epitope
aa 41-58; Bric163, recognize an intracellular epitope; TM region aa 73-95),
and against GPC (Bric4, extracellular epitope aa 16-23; Bric10, extracellu-
lar epitope aa 1-6; BGRL 100, intracellular epitope aa 112-128; TM region,
59-81).26 Anti-GPB was from Sigma Chemical. Monospecific rabbit
polyclonal sera against CR1, C3, and C8 were produced in-house.
RAM/IgG–fluorescein isothiocyanate (FITC) was from Dako (High Wy-
combe, Bucks, UK), GAM/IgG-HRP was from Bio-Rad (Hemel, Hemp-
stead, UK), and GAR/IgG-FITC was from Sigma Chemical.
Venom
Laboratório de Imunoquı́mica (Instituto Butantan, São Paulo, Brazil)
provided Loxosceles intermedia Mello-Leitão spiders. The venom was
obtained by electrostimulation by the method of Bucherl,27 with slight
modifications. Briefly, electrical stimuli of 15 to 20 V were repeatedly
applied to the spider sternum, and the venom drops were collected with a
micropipette, vacuum dried, and stored at 220°C. Stock solutions were
prepared in PBS at 1 mg/mL. Toxins (P1, P2, and P3) from L. intermedia
were purified by Superose 12-gel filtration followed by reverse-phase
high-performance liquid chromatography using a Wide-Pore Butyl C4
column (Pharmacia, Uppsala, Sweden) as described.10 The protein content
of the samples was evaluated by the Lowry method.28
Production of rabbit antiserum against F35
Adult rabbits were injected intradermally with 500 ng of F35 (unfraction-
ated P1, P2, P3)9 absorbed to Al(OH)3. Injections were repeated 4 times at
weekly intervals. Blood samples were collected 1 week after the last
injection, and the serum was stored at 220°C.
Normal human serum and erythrocytes
Human blood was obtained from healthy donors. Blood samples drawn to
obtain sera were collected without anticoagulant and allowed to clot for 2
hours at room temperature, and the normal human serum (NHS) was stored
at 280°C. C8-depleted human serum (C8d-HS) was obtained by the
passage of NHS over an mAb anti-C8 Sepharose 4B column. Blood
samples drawn to obtain E for subsequent use as target cells were collected
in anticoagulant (Alsever old solution: 114 mmol/L citrate, 27 mmol/L
glucose, 72 mmol/L NaCl, pH 6.1).
Treatment of E with Loxosceles venom proteins
E were washed and resuspended at 2% in VBS21 and incubated with whole
venom or purified fractions for 30 minutes at 37°C. Control samples were
incubated with VBS21. Purified fractions did not induce spontaneous lysis
of the cells. The cells were washed 5 times, resuspended to the original
volume in VBS21, analyzed in a hemolysis assay, and prepared for flow
cytometry or Western blot analysis. For Western blotting, E ghosts were
prepared by lysis of E in water. Ghosts were pelleted by centrifugation
(14 000g for 20 minutes at 4°C) and washed with water.
Treatment of E with neuraminidase
One milliliter 2% HuE suspension was incubated with 0.2 U of neuramini-
dase for 1 hour at 37°C. Cells were washed 5 times and resuspended to the
original volume in VBS21 and assayed as described.
Treatment of purified glycophorin A with L. intermedia venom
toxins
Purified GPA (2 µg) was incubated with VBS21, the L. intermedia venom,
or the purified toxins P1, P2, and P3 (2 µg each) in a total volume of 20 µL in
VBS21 at 37°C for 60 minutes. Samples were run on sodium dodecyl
sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by
Western blotting.
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BLOOD, 15 JANUARY 2000 • VOLUME 95, NUMBER 2 LOXOSCELES VENOM-INDUCED CLEAVAGE OF GLYCOPHORIN 685

Hemolysis assays glycophorin mAb (1 µg/mL) for 1 hour at room temperature. Membranes
were washed 3 times with PBS/0.05% Tween 20 for 10 minutes and
One hundred microliters 2% E pretreated with Loxosceles venom, purified
incubated with GAM/IgG-HRP 1/3000) in PBS/BSA 1% for 1 hour at room
toxin P1, P2, or P3, and neuraminidase or VBS21 were mixed with 100 µL
temperature. After they were washed 3 times with PBS/0.05% Tween 20 for
NHS (1/2 in VBS21). Background or total cell lysis was evaluated by
10 minutes and twice with PBS, blots were developed using Supersignal
incubation of E with VBS21 or H2O respectively. After incubation for 1
chemiluminescent substrate (Pierce) and Kodak X-ray film (Eastman
hour at 37°C, unlysed cells were spun down; the absorbance of the
Kodak, Rochester, NY).
supernatant was measured at 541 nm and expressed as a percentage of lysis.
Mean and SD were determined from duplicate samples. E and NHS were Pretreatment of E with protease inhibitors
always from the same donor.
E were incubated with 10 mmol/L EDTA, 5 mmol/L, 1,10-phenanthroline,
Calcein-AM loading of E 1 mmol/L PMSF, or buffer for 30 minutes on ice. Venom or toxins were
added and incubated for 30 minutes at 37°C. Cells were washed 3 times and
E were washed, resuspended at 2% in VBS21 containing 1/200 dilution of analyzed for C susceptibility or GP expression by flow cytometry.
calcein-AM (1 mg/mL stock in dimethyl sulfoxide), and incubated for 30
minutes at 37°C. The cells were washed twice and resuspended to the
original volume in VBS21.
Results
Transfer of hemolysis-inducing activity
C activation induced by L. intermedia venom toxins
Samples of buffer-, venom-, and toxin-treated E were mixed with the same
volume of calcein-loaded E suspension or with buffer and incubated for 30 To assess the ability of L. intermedia venom to induce C-dependent
minutes at 37°C. After this period, cells were washed once, resuspended hemolysis, E were incubated with 10 µg/mL L. intermedia venom
with VBS21, and analyzed for autologous C lysis susceptibility as described or purified P1, P2, or P3 toxin and were assessed for the
above. Final lysis was measured spectrophotometrically at 541 nm and susceptibility to lysis by human C. As shown in Figure 1A, the L.
fluorometrically by measuring the calcein fluorescence of the supernatants intermedia venom and the pure proteins P1 and P2, but not P3,
in a Denley-Wellfluor fluorometer with the excitation filter at 488 nm and were able to render E susceptible to lysis by autologous C. A
the emission filter at 530 nm. Percentage lysis for each sample was similar level of C susceptibility was obtained after incubation of the
calculated as specific hemoglobin release/total hemoglobin and as specific
calcein release/total calcein loading. Mean and SD were determined from
duplicate samples.

Nucleated cells
The K562 (erythroblast), U937 (promonocyte), and Jurkat (T-cell) cell lines
were obtained from the European Collection of Animal Cell Cultures
(Porton Down, Salisbury, UK). Cells were cultured in RPMI-1640 medium
supplemented with 10% fetal calf serum, 4 mmol/L glutamine, 2 mmol/L
sodium pyruvate, 100 IU/mL penicillin, and 100 IU/mL streptomycin at
37°C and 5% CO2.

Treatment of nucleated cells with P2 toxin

Log-phase nucleated cells were harvested, washed 3 times in PBS, and
resuspended in VSB21 at 107/mL. The cells were treated with 10 µg/mL P2
toxin for 30 minutes at 37°C. Control samples were incubated with VSB21.
The cells were washed 5 times, resuspended to the original volume in
21
VSB , and prepared for analysis by flow cytometry.

Flow cytometry
E (50 µL 2%) or 50 µL 106 nucleated cells were incubated for 30 minutes at
4°C with 50 µL of 1 µg/mL anti-C regulators or anti-glycophorin mAb or
with rabbit antiserum recognizing P1, P2, and P3 (F35; diluted 1:250) in
FACS buffer. After they were washed, cells were incubated with RAM/IgG-
FITC or GAR/IgG-FITC for 30 minutes at 4°C. Cells were washed and
fixed in FACS buffer containing 1% paraformaldehyde and were analyzed
by flow cytometry (FACScalibur; Becton Dickinson, San Jose, CA).

Analysis of deposition of C components

E treated with spider toxins was incubated with C8-depleted serum (1/10 in
VBS21, 30 minutes, 37°C), washed, and processed for flow cytometry using
rabbit polyclonal anti-C component sera diluted 1/100 and was followed by
GAR/IgG-FITC as described above.
Figure 1. Induction of C-susceptibility by L. intermedia venom toxins. (A)
Electrophoresis and Western blot analysis C-dependent hemolysis of E after incubation with VBS21 (C), with 10 µg/mL of L.
intermedia venom (V), or with purified toxin P1, P2, or P3. (B) C3b deposition after
E ghosts (10 µL) or purified GPA samples (1 µg) were solubilized in a
treatment of E, followed by incubation with C8-depleted serum. Deposition was
nonreducing sample buffer and run on 12% SDS-PAGE.29 Gels were measured using polyclonal sera against C3 and analysis by flow cytometry. Results
stained with silver30 or blotted onto nitrocellulose. After transfer, the are representative of 3 different experiments carried out in duplicate and represented
membranes were blocked with PBS/BSA 1% and incubated with anti- as mean 6 SD.
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686 TAMBOURGI et al BLOOD, 15 JANUARY 2000 • VOLUME 95, NUMBER 2

cells with neuraminidase (data not shown). To assess the effect of

the toxins on C3 deposition, toxin-treated E were incubated with
C8-depleted human serum and analyzed by flow cytometry for the
deposition of C3b. Figure 1B shows an increased deposition of C3b
on the E treated with venom, P1, or P2, but not with P3 or buffer.

Effect of L. intermedia toxins on membrane-bound regulators

of C
To assess whether the increased susceptibility to human C was
caused by interference of the toxins with membrane regulators of
C, E were analyzed for the expression of DAF, CR1, and CD59 by
flow cytometry. No change in expression of any of the regulators
was observed after incubation of E with whole venom or any of the
purified toxins (data not shown).

Removal of glycophorin from E induced by L. intermedia

venom toxins
Although DAF, CR1, and CD59 are powerful inhibitors of
C-mediated lysis, the abundantly expressed, heavily glycosylated
E-membrane proteins known as glycophorins also contribute
substantially to C resistance.19-24 E, incubated with L. intermedia
toxins, were analyzed for the expression of GPA and GPC by flow
cytometry. A large reduction in the binding of anti-GPA (Bric256)
and anti-GPC (Bric4, Bric10) antibodies recognizing extracellular
epitopes close to the membrane was observed after treatment of E
with venom, P1, or P2 (Figure 2). The disappearance of these
epitopes was associated with the incorporation of the toxins into E,
as detected by antibody F35 (Figure 2). These data show also that
P3 does not incorporate into E, which may account for its lack of
toxicity.

Analysis of the cleavage of glycophorins induced by active

venom toxins
To assess whether removal of the GP epitopes resulted from the
cleavage of GP or from complete extraction, E were treated with
venom toxins, and, after washing, ghosts were prepared by
hypotonic lysis. E ghosts were analyzed by Western blot using
mAb recognizing intracellular and extracellular epitopes of GPA,
GPB, and GPC. As shown in Figure 3, incubation of E with whole
Figure 2. Loxosceles venom components incorporate into E and cause loss of
expression of glycophorins. E were treated with buffer (C), L. intermedia venom,
P1, P2, or P3 toxins (10 µg/mL each), and analyzed for the expression of GPA and
GPC by flow cytometry using the antibodies Bric256 (GPA) and Bric4 and Bric10
(GPC). The ability of the toxins P1, P2, and P3 to insert into the E surface was
analyzed using a monospecific polyclonal rabbit serum (F35) against the toxins.
Results are representative of 3 different experiments and are expressed as mean of
triplicates 6 SD.
Figure 3. Cleavage fragments of GPA, GPB, and GPC remain on E ghosts after
treatment with L. intermedia venom toxins. E were treated with VBS21 (C) or with
10 µg/mL of L. intermedia venom (V) or purified toxins P1, P2, or P3. E ghosts were
prepared and run on 12% SDS-PAGE under nonreducing conditions (A to D) or were
stained with silver (E). Western blots: A, GPA extracellular epitope; B, GPA
intracellular epitope; C, GPB extracellular epitope; D, GPC intracellular epitope.
venom or toxins P1 and P2 resulted in the cleavage of all
glycophorins.
Western blot analysis revealed 2 bands in GPA, a monomeric
form of 41 kd and a dimer with an Mr of 82 kd. Using antibodies
that recognize an extracellular epitope in GPA, Western blotting
showed that on incubation with venom toxins, a nearly complete
loss of this epitope was induced (Figure 3A). Using an mAb
recognizing an intracellular epitope of GPA, 2 bands again were
observed, but on incubation of the E with venom toxins, a large
reduction in Mr of the bands was observed (Figure 3B). This shows
that the intracellular epitope was retained in the E and that the loss
of the extracellular epitope was caused by the cleavage of GPA
rather than by extraction of the whole molecule. In this case, venom
or P1 and P2 treatment resulted in multiple GPA fragments of GPA
ranging from 19 to 27 kd. The occurrence of multiple bands may
have been caused by the cleavage of GPA at multiple sites. Some
bands may also have represented dimers of cleaved GPA fragments.
GPB ran as a single band on Western blotting and was detected
using an mAb against an extracellular epitope (Figure 3C). This
epitope completely disappeared on incubation of E with venom
toxins. An mAb against an intracellular epitope of GPB was
unavailable.
GPC runs as a monomer (Mr, 44 kd) on SDS-PAGE (Figure
3D). The N-terminal truncated form of GPC, GPD is also detected
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BLOOD, 15 JANUARY 2000 • VOLUME 95, NUMBER 2 LOXOSCELES VENOM-INDUCED CLEAVAGE OF GLYCOPHORIN 687

as a monomer of 27 kd in the gel. Using an mAb recognizing an

intracellular epitope of GPC, it was shown that on the incubation of
E with venom, P1, or P2, but not P3, these bands disappeared and a
single band with an Mr of 20 kd was observed. This band
represented the transmembrane and cytoplasmic fragment of
GPC/GPD retained in the ghosts (Figure 3D). These data demon-
strate that glycophorins are cleaved and are not extracted on venom
toxin incubation.
Silver staining of the gels showed equal loading in all lanes,
indicating that the absence of GP bands on Western blots did not
result from the loss of venom-treated cells during ghost preparation
(Figure 3E). Silver staining also showed the loss of some bands
with Mr 40 to 42 kd and 80 to 82 kd on venom toxin-treated E
(indicated by arrows), which most likely represented GPA (Figure
3E). Because of their abundance in E and of heavy glycosylation,
GPs are strongly stained by silver on SDS-PAGE. B
GPA cleavage is not caused by a direct proteolytic action of L.
intermedia toxins
Toxins P1 and P2 have been shown only to have sphingomyelinase
activity.10 To analyze whether GP cleavage resulted from direct
proteolytic action of toxins on glycophorins, purified GPA was
incubated with whole venom or purified toxins. Samples were
submitted to nonreducing SDS-PAGE, followed by Western blot
analysis, using the mAb recognizing the intracellular domain of
GPA. Figure 4 shows that cleavage of GPA was not induced.
Increases in incubation time or toxin content of the samples did not
result in any alteration in the mobility and banding patterns of GPA Figure 5. P2 incorporates into E and causes dose-dependent cleavage in
(data not shown). These results show that P1 and P2 are not specific glycophorin expression and hemolytic susceptibility. (A) E were treated with
glycophorin proteases, and they suggest that the GP cleavage various concentrations of P2 and analyzed by flow cytometry using the mAb for GPA
(h), GPB (V), and GPC (n) or the polyclonal serum F35 against P2 (s). Cells were
process is caused by the activation of an endogenous E protease also subjected to C in a hemolysis assay (d). (B) E ghosts obtained from samples
induced by P1 and P2 toxins. described above and were prepared and analyzed by Western blot using the
monoclonal antibody against the intracellular portion of GPA (Bric163).
P2 incorporation correlates with glycophorin cleavage and C
susceptibility
susceptibility. P2 toxin became incorporated into E in a dose-
E were treated with increasing concentrations of P2 toxin and were dependent manner and reached a maximal incorporation at approxi-
analyzed by flow cytometry for the expression of GPA, GPB, and mately 20 µg/mL (equivalent to the addition of 3.4 3 106 mol-
GPC, for the incorporation of P2 into the E membrane, and for C ecules per cell; Figure 5A). Concomitant with the incorporation of
P2, a decrease in the expression of GPA, GPB, and GPC and an
increase in C susceptibility were observed. The disappearance of
the GPB epitope was slow, possibly reflecting a relative resistance
of GPB to cleavage. Western blot analysis of E ghosts showed that
GPA was already hydrolyzed in the presence of 2.5 µg/mL of the
toxin (Figure 5B). Complete fragmentation of GPA was achieved
within 30 minutes in E preparations treated with 10 µg/mL of the
toxin P2 (Figure 5B). In this experiment, only 2 fragments of GPA
were obtained, likely representing monomeric and dimeric forms of
the transmembrane portion and cytoplasmic tail, suggesting that in
this experiment cleavage was induced at only 1 site. These
experiments showed a positive correlation between the cleavage of
GPA, GPB, and GPC and increased C susceptibility induced by
increasing amounts of E-bound P2 toxin.

Transfer of hemolysis-inducing activity

Figure 4. Loxosceles venom and purified toxins do not cleave purified
glycophorin A. Purified GPA (2 µg) was incubated with buffer (C) or with 2 µg of L.
intermedia venom (V), P1, P2, or P3 toxins for 60 minutes at 37°C. Samples were
separated by SDS-PAGE under nonreducing conditions (12% gel) and analyzed by
Western blot using the monoclonal antibody Bric163 .
Although the bite of the L. intermedia spider results in the secretion
of only a fraction of a microliter of venom containing not more than
30 µg toxin, in incidents of systemic effects extensive intravascular
hemolysis is observed.1-8 The low amount of toxin injected could
not account for the large number of erythrocytes lysed unless the
toxins can transfer from 1 cell to the other in vivo and hence have
an effect on many erythrocytes. To test this hypothesis, venom
 From www.bloodjournal.org by guest on May 25, 2017. For personal use only.
688 TAMBOURGI et al
toxin-treated E were mixed with untreated E. After incubation, the
mixtures were assessed for their susceptibility to C. To distinguish
between lysis of E incubated with venom toxins and the freshly
added untreated E, the untreated E were labeled with the fluores-
cent dye calcein. Lysis of the untreated E could then be measured as
the release of the entrapped calcein, whereas lysis of the treated and
untreated E was measured as the release of hemoglobin (Hb).
Venom-treated and untreated E were mixed 1:1 so that an Hb
release of more than 50% would indicate that untreated E were also
lysed. Figure 6A shows the hemolysis of the total E. Nearly 100%
release of hemoglobin was obtained at the highest dose of P2,
demonstrating that the erythrocytes that had not been incubated
with toxins were lysed. This was confirmed by the observation that
nearly 100% of the entrapped calcein could be released from the E
that had not been treated with toxins (Figure 6B). The cells were
Figure 6. Transfer of hemolysis-inducing and glycophorin-cleaving activity. E
were treated with increasing concentrations of P1 (d), P2 (s), or L. intermedia venom
(*), washed, and incubated with the same volume and number of calcein-AM loaded
E or buffer. After 30 minutes of incubation at 37°C, cells were analyzed for autologous
C lysis (A, B) or for the expression of GPC (C). (A) Total hemoglobin release induced
by NHS determined spectrophotometrically at 541 nm. (B) Release of entrapped
calcein determined fluorometrically. [C] E samples incubated with buffer or with P2
(2.5 and 5.0 µg/mL) were incubated with the same volume of untreated E (h) or with
buffer (j) for 30 minutes at 37 °C and analyzed by flow cytometry for expression of
GPC using the monoclonal antibody Bric4. Results are expressed as the percentage
of fluorescence of untreated E.
BLOOD, 15 JANUARY 2000 • VOLUME 95, NUMBER 2
also analyzed by flow cytometry for the cleavage of GPA, and, as
shown in Figure 6C, in the P2-treated E and in the mixture of
P2-treated and untreated E, a similar pattern of reduction of GPA
expression was induced. These results show that hemolysis-
inducing activity can be transferred to a new erythrocyte popula-
tion and that this phenomenon can explain the extent of the
systemic hemolysis observed after envenomation. The sensitivity
of detection of P2 by flow cytometry was not adequate to observe
the actual transfer of the toxin.
Removal of extracellular domains of GPC from the surface of
nucleated cells
Although GPA is predominantly expressed on cells of the erythro-
cyte lineage, GPC is expressed on a wide range of cells. To
establish whether glycophorin cleavage could occur in cells other
than E, 3 different nucleated cell types—K562 (erythroid), U937
(myeloid), and Jurkat (lymphoid)—were incubated with L. interme-
dia toxins and analyzed for the expression of GPC by flow
cytometry. Figure 7 shows that venom treatment induced the loss of
GPC from all these cells.
Inhibition of venom and toxins
Cleavage of glycophorins was shown not to be a direct action of the
Loxosceles toxins (Figure 4). The hypothesis that a membrane-
bound protease was involved was investigated. Many of the known
membrane-bound proteases that release cell-bound molecules are
metalloproteinases. The effect of EDTA (binding of divalent
cations), 1,10 phenanthroline (specific for metalloproteinase), and
PMSF (inhibitor of serine proteases) on the ability of the Loxosce-
les venom and purified toxins to cleave glycophorins and induce C
susceptibility was assayed. Both EDTA and 1,10 phenanthroline
inhibited the cleavage and induction of C susceptibility by P2
(Figure 8). Previously, we showed that Ca11 is necessary for the
sphingomyelinase activity of the Loxosceles toxins and that it can
be inhibited by EDTA.10 In this study, we showed that toxin binding
to the E membrane is also partially blocked by EDTA. However,
1,10 phenanthroline did not have an effect on toxin binding but
only prevented glycophorin cleavage and the induction of C
susceptibility. Given the known properties of 1,10 phenanthroline,
these data suggest that a metalloproteinase is activated that is
responsible for the cleavage of glycophorin. Results obtained with
whole venom or P1 were similar to that obtained with P2 (data not
shown).
Discussion
The bite of spiders of the genus Loxosceles can induce various
biologic effects, including dermonecrosis and C-dependent hemoly-
sis.1-8 The aim of this study was to elucidate the mechanism of
action of L. intermedia venom, its active toxins P1 and P2, and in
particular the mechanism of induction of C susceptibility. We have
previously shown that in vitro hemolysis of erythrocytes, induced
by Loxosceles venom, is accomplished by activation of the
alternative pathway of C.9 In this study we did not observe an effect
of Loxosceles venom and purified toxins on the expression of
C-regulators CD59, DAF, or CR1, eliminating loss of C regulators
as a cause of hemolysis. However, Loxosceles venom and purified
toxins P1 and P2 efficiently induced the loss of GPA, GPB and
GPC (Figures 2 and 3). Using mAbs specific for intracellular and
extracellular epitopes of GPA, GPB, and GPC, we showed that
 From www.bloodjournal.org by guest on May 25, 2017. For personal use only.

BLOOD, 15 JANUARY 2000 • VOLUME 95, NUMBER 2 LOXOSCELES VENOM-INDUCED CLEAVAGE OF GLYCOPHORIN 689

neous activators of C, resulting in massive hemolysis often

followed by kidney failure.1-8 Erythrocytes express 1 to 5 3 104
copies of CD59,31,32 4 3 103 copies of DAF,33 and 1 to 8 3 103
copies of CR1.34 Glycophorins are more abundant (molecules per
E: GPA, 106; GPB, 2.5 3 105; GPC, 5-12 3 104),20 and on a
mole-to-mole basis the C-regulatory molecules are likely to be
more effective than the glycophorins.
Deficiencies of glycophorin have been described, but hemolytic

Figure 7. P2 toxin induces cleavage of GPC from nucleated cells. K562, U937,
and Jurkat cells were incubated for 30 minutes with buffer (thick line) or with 10 µg of
P2 toxin (thin line), washed and stained for flow cytometry using the monoclonal
antibody Bric4 against GPC. Background fluorescence (shaded histogram). Results
are representative for 2 different experiments.

glycophorins are cleaved extracellularly (Figure 3). Glycophorin

cleavage was accompanied by the induction of C susceptibility.
Glycophorin cleavage was only observed when the active compo-
nent of the venom or the purified toxins P1 and P2 bound to the
membrane. The inability of the venom component P3, despite its
high homology with P1 and P2, to induce C-dependent hemolysis
is here shown to result from its inability to bind the erythrocyte
membrane.
GPA is the major membrane sialoglycoprotein of human E, and
it represents a typical example of a transmembrane glycoprotein.
The main functional role of GPA is thought to be in retaining E
structural integrity. Purified glycophorins inhibit C-mediated ly-
sis,19,21,22 and the removal of sialic acid makes E activators of the
alternative pathway of C.23,24 Our results suggest that a major
functional role of glycophorin is in protection from homologous C
and that glycophorin may be more important in this respect than Figure 8. Chelating agents inhibit Loxosceles venom-induced cleavage of
DAF or CD59. In support of this suggestion, PNH E, which lack glycophorins and induction of C susceptibility. E were incubated with EDTA (10
both DAF and CD59, exhibit only low-grade hemolysis in vivo.14-17 mmol/L), 1,10-phenanthroline (5 mmol/L), or PMSF (1 mmol/l). Loxosceles venom or
toxins were added and incubated 30 minutes 37°C. Cells were washed and analyzed
PNH E are not spontaneous activators of C, and marked lysis is
for C susceptibility (A), expression of GPC using mAb Bric4 (B), incorporation of
only observed on in vitro activation of C by acidification of the toxins using Ab F35 (C). Results are mean 6 SEM of experiments carried out in
serum. In contrast, on Loxosceles envenomation, E become sponta- triplicate.
 From www.bloodjournal.org by guest on May 25, 2017. For personal use only.

690 TAMBOURGI et al BLOOD, 15 JANUARY 2000 • VOLUME 95, NUMBER 2

syndromes have not been observed in patients,20,35-37 probably
because not all the glycophorin species are missing in these
syndromes. However, it cannot be excluded that in Loxosceles
envenomation, factors other than glycophorin removal affect the E
susceptibility to C. Change in lipid composition has been shown to
have an effect on C susceptibility, probably because of a change in
efficiency of MAC binding.25 Increased accessibility of the mem-
brane because of removal of the bulky glycophorin molecules
might also contribute to the increase in complement susceptibility.
However, Loxosceles toxins do not increase the susceptibility to
lysis by other pore formers such as perforin or melittin (Tambourgi
et al, unpublished observations). Extensive hemolysis of E also
occurs in the hemolytic uremic syndrome.38-40 In this disease
various factors have been identified, including a deficiency in
factor H40 and desialylation of glycophorin by bacterial neuramini-
dases.41 Another common form of HUS occurs in the presence of
fH, which is induced by verotoxins produced by certain bacteria.42
These toxins bind to a glycoceramide43 and have a substrate
specificity similar to that of Loxosceles toxins. It would be of
interest to examine the effects of these toxins on glycophorin
expression. The only sphingomyelinases functionally similar to
Loxosceles toxins are some bacterial phospholipases D (PLD) from
Corynebacterium pseudotuberculosis44 and Arcanobaterium haemo-
lyticum. This PLD also shows 24% to 30% homology with the first
30 amino acids of the Loxosceles toxins,10 and it has a similar
molecular weight, pI, and substrate specificity.44 The purified PLD
from C. pseudotuberculosis can also cause hemolysis and kidney
failure in lambs.45 It is unknown whether complement is involved
in this process. Phospholipase D from, for example, cabbage and
Streptomyces pyogenes does not induce C susceptibility (data not
shown), whereas sphingomyelinase C causes E lysis in the absence
of C.
Cleavage of glycophorin could not be reproduced using purified
toxins and GPA, inducing us to investigate whether hydrolysis of
sphingomyelin by the spider toxins activate an endogenous prote-
ase responsible for the cleavage of glycophorins. Membrane-bound
secretases, also called sheddases or membrane-protein convertases,
are responsible for the cleavage of many membrane-bound pro-
teins.46 Most secretases depend on metal ions, and we show that the
cleavage of glycophorin and the induction of C susceptibility were
inhibited by EDTA and 1,10-phenanthroline. Although EDTA also
inhibited the binding of the toxins to the cells, in the presence of
1,10-phenanthroline binding occurred but the toxin-induced glyco-
phorin cleavage was inhibited. 1,10 Phenanthroline binds Zn21 and
is a powerful inhibitor of metalloproteinase, including those of the
ADAMs family.47 We suggest that venom proteins, through their
sphingomyelinase activities, alter the membrane environment and
membrane fluidity, causing activation of an as yet unknown
metalloproteinase. Recently metalloproteinase activity has been
detected in Loxosceles venom.48 This metalloproteinase with Mr 35
kd had substrate specificity for fibronectin as shown in a zymogram
assay, but it has not been purified and characterized in more detail.
It is unlikely that this protein is the same molecule as our purified
toxins P1 and P2, which lack direct proteolytic activity toward
glycophorin. A few E metalloproteinase have been characterized,
but no natural substrates have been identified.49-51 The identity of
the metalloproteinase involved in glycophorin cleavage remains to
be elucidated.
The glycophorins are all similar in topology, but they are
distinct in sequence.20 Although GPA and GPC are cleaved on
treatment with venom toxins, no obvious sequence homology in the
extracellular regions of GPA and GPC is observed. Sheddases often
have a broad specificity and require only 1 or 2 specific amino acids
at the site of cleavage. We did not observe a change in expression of
any of the C regulators or on band 3 (data not shown), and there
were no gross changes in E-protein pattern on SDS-PAGE, but it
remains possible that other membrane-bound molecules are also
cleaved.
The hemolysis-inducing and glycophorin-cleavage activity was
transferred from treated E to a new E population (Figure 6). This
transfer phenomenon explains the extent of the systemic hemolysis
observed after envenomation. This situation is not unique in that
the transfer of sphingomyelinase activity between cells has been
reported.52
In contrast to GPA and GPB, which appear late during erythroid
development, at the proerythroblast stage, GPC is already present
on erythroid progenitors and can be detected on leukocytes. Venom
toxins caused a nearly complete removal of GPC expressed on the
human cell lines K562 (erythroblast), U937 (promonocyte), and
Jurkat (T cell), demonstrating that the toxins can induce activation
of proteases in nucleated cells. The observation that metalloprotein-
ase in nucleated cells is activated by venom proteins may help
explain the local effects of dermonecrosis.
In conclusion, we present a mechanism through which Loxosce-
les venom renders E susceptible to lysis by C, which explains the
extent of hemolysis observed in patients after envenomation and
shows that it is possible to inhibit a biologic effect of this harmful
venom. The discovery of the involvement of metalloproteinase in
the toxicity of the Loxosceles venoms is novel and of obvious
therapeutic significance given the availability of metalloproteinase
inhibitor drugs. Our unpublished data show a similar activity of
purified toxins from at least 2 other Loxosceles species. The
mechanism by which the spider toxins induce activation of
metalloproteinase on E—identification of the metalloproteinase
responsible for glycophorin cleavage and testing of a therapeutic
strategy—are the subjects of further study.

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2000 95: 683-691

Loxosceles intermedia spider envenomation induces activation of an

endogenous metalloproteinase, resulting in cleavage of glycophorins from
the erythrocyte surface and facilitating complement-mediated lysis
Denise V. Tambourgi, B. Paul Morgan, Rute M. G. de Andrade, Fábio C. Magnoli and Carmen W. van
den Berg

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